: Posttranscriptional modifications in ribosomal ribonucleic acids (rRNAs) are difficult to identify and their function is unknown. Our knowledge of the functional role of these posttranscriptional modifications is limited largely by the lack of methods for their routine identification. The long-term goal of our research has been and continues to be to develop appropriate mass spectrometric approaches to characterize the structure of the ribosome in terms of RNA. The goal of this funding period will be to extend our previous improvements in mass spectrometric methods for identifying posttranscriptional modifications in rRNA in order to permit the structural interactions between (modified) nucleosides in rRNA and ribosomal proteins to be characterized. We will develop new, improved mass spectrometry (MS) methods to characterize ribonucleoprotein (RNP) interactions within the ribosome. We will use matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to identify cross-links between RNA and ribosomal proteins. We will extend our current developments using RNase H to digest rRNAs to focus on particular regions of the ribosome as a means of simplifying the determination of protein:nucleic acid cross-links. In particular, we will use this new approach to focus on areas of rRNA known to contain posttranscriptionally modified nucleosides to ascertain the structural significance of such modifications. We will use limited proteolysis with MALDI-MS to characterize ribosome organization, topology and proteins suspected of directly interacting with rRNA. Limited proteolysis, cross-linking and analysis by MALDI-MS and liquid chromatography mass spectrometry will be used to identify sites of interaction between ribosomal proteins and rRNAs suspected of being important in ribosome assembly. As pseudouridine is the most common posttranscriptionally modified nucleoside, we propose to develop an improved method for identifying this modification and analyzing its role in rRNA:rRNA and rRNA:protein interactions. Furthermore, we will apply advances in method developments in proteomics to initiate studies aimed at characterizing the structural interactions of bacterial ribosomes as a function of cell growth conditions. These latter studies will open up new avenues of research in characterizing the dynamic nature of the ribosome. In addition, this research will facilitate future studies that will help determine how ribosomes can carry.out the task of translation with amazing speed and accuracy.